Copines are highly conserved calcium-dependent membrane-binding proteins found in numerous diverse eukaryotic organisms. The wide array of organisms ranging from single-celled organisms to humans in which copines are found suggest that copines carry out fundamental functions important in most eukaryotic cells. A growing body of evidence indicates that copines act as regulatory proteins in signaling pathways. The model organism Dictyostelium discoideum has six copine genes (cpnA-cpnF) and provides an ideal system for studying copine function. Dictyostelium can live independently as single-celled amoebae, but when placed in starvation conditions, single amoeba signal each other to first aggregate and then differentiate into cells that form a multicellular fruiting body. This simple developmental program in Dictyostelium is widely used to study not only development, but also several basic cell processes including chemotaxis-mediated cell motility, signal transduction, programmed cell death, and cell differentiation. Previous studies showed that one of the copine proteins in Dictyostelium, CpnA, is required for normal development. The overall goal of the previous grant award was to correlate our findings on cpnA- cells during developmental processes (aim 1) and the identification of the target proteins of CpnA (aim 2) to generate more specific hypotheses about the function of CpnA. Significant progress was made on both of these specific aims and data collected during the previous grant award period has led to two new hypotheses about the function of CpnA. These two hypotheses form the two specific aims of this renewal proposal: 1) Determine if CpnA is a negative regulator of the cAMP phosphodiesterase, RegA and 2) Determine if CpnA is a negative regulator of actin filament polymerization. This grant proposal describes experiments using biochemical, genetics, and microscopy approaches to test these hypotheses. The results of these experiments will make a significant impact on this field by being the first to define a mechanistic role for a copine protein. Human copines have been implicated to play a role in cancer by regulating signaling pathways involving the ErB2 receptor and in immune responses by regulating signaling pathways involving the TNF (tumor necrosis factor) receptor. Increasing our scientific knowledge of how copines function could lead to a better understanding of cancer and immune cell-related diseases.